Previous work supported by this RO1 has suggested that the initiation of pancreatic tumorigenesis may involve cells with an undifferentiated precursor phenotype. The long-term goal of this research program remains the identification and eventual therapeutic manipulation of exocrine precursor cells as the probable "cell of origin" for pancreatic ductal cancer. In developing mouse and zebrafish pancreas, exocrine precursor cells are characterized by expression of a panel of markers, including nestin, hesl1 and ptf1-p48. Additional data now suggest that these cells also express vertebrate orthologs of Drosophila musashI an RNA binding protein known to be required for maintenance of precursor phenotypes in the developing nervous system. In developing pancreas, musashi is expressed in undifferentiated pancreatic epithelium, but not in differentiated pancreatic cell types. These observations generate the hypothesis that musashi is also required to maintain an undifferentiated precursor population in developing pancreas, and that undifferentiated musashi-positive epithelial cells represent direct precursors for the exocrine lineage. To test these hypotheses, the following ISpecific Aims will be pursued: First, to characterize temporal and spatial patterns of musashi expression in developing mouse and zebrafish pancreas, and to determine gain-of-function and loss-of-function phenotypes associated with altered musashi expression; Second, to determine the differentiation potential of! musashi-expressing precursor cells in developing mouse pancreas using formal lineage tracing techniques; and Third, to identify and characterize target RNA's bound by musashi in developing mouse and zebrafish pancreas, anticipating that these targets will represent positive regulators of pancreatic epithelial differentiation. Together, these studies will provide important information regarding exocrine precursor populations in developing pancreas. The pursuit of these studies in both mouse and zebrafish model systems will allow development of insights not easily ascertained using any single approach. By delineating mechanisms by which an undifferentiated precursor pool is maintained in developing pancreas, these studies will further advance our understanding of pancreatic development, pancreatic regeneration and pancreatic cancer.